1. Field of the Invention
The present invention relates to a method of improving processes using pectinase enzymes with noble gases.
2. Description of the Background
The ability of the noble gases helium (He), neon (Ne), argon (Ar), krypton (Kr), xenon (Xe) and radon (Ra) to enter into chemical combination with other atoms is extremely limited. Generally, only krypton, xenon and radon have been induced to react with other atoms, which are highly reactive such as fluorine and oxygen, and the compounds thus formed are explosively unstable. See Advanced Inorganic Chemistry, by F. A. Cotton and G. Wilkinson (Wiley, Third Edition). However, while the noble gases are, in general, chemically inert, xenon is known to exhibit certain physiological effects, such as anesthesia. Other physiological effects have also been observed with other inert gases such as nitrogen, which, for example, is known to cause narcosis when used under great pressure in deep-sea diving.
It has been reported in U.S. Pat. No. 3,183,171 to Schreiner that argon and other inert gases can influence the growth rate of fungi and argon is known to improve the preservation of fish or seafood. U.S. Pat. No. 4,946,326 to Schvester, JP 52105232, JP 80002271 and JP 77027699. However, the fundamental lack of understanding of these observations clearly renders such results difficult, if not impossible, to interpret. Moreover, the meaning of such observations is further obscured by the fact that mixtures of many gases, including oxygen, were used in these studies. Further, some of these studies were conducted at hyperbaric pressures and at freezing temperatures. At such high pressures, it is likely that the observed results were caused by pressure damage to cellular components and to the enzymes themselves.
For example, from 1964 to 1966, Schreiner documented the physiological effects of inert gases particularly as related to anesthetic effects and in studies relating to the development of suitable containment atmospheres for deep-sea diving, submarines and spacecraft. The results of this study are summarized in three reports, each entitled: "Technical Report. The Physiological Effects of Argon, Helium and the Rare Gases," prepared for the Office of Naval Research, Department of the Navy. Contract Nonr 4115(00), NR: 102-597. Three later summaries and abstracts of this study were published.
One abstract, "Inert Gas Interactions and Effects on Enzymatically Active Proteins," Fed. Proc. 26:650 (1967), restates the observation that the noble and other inert gases produce physiological effects at elevated partial pressures in intact animals (narcosis) and in microbial and mammalian cell systems (growth inhibition).
A second abstract, "A Possible Molecular Mechanism for the Biological Activity of Chemically Inert Gases," In: Intern. Congr. Physiol. Sci., 23rd, Tokyo, restates the observation that the inert gases exhibit biological activity at various levels of cellular organization at high pressures.
Also, a summary of the general biological effects of the noble gases was published by Schreiner in which the principal results of his earlier research are restated. "General Biological Effects of the Helium-Xenon Series of Elements," Fed. Proc. 27:872-878 (1968).
However, in 1969, Behnke et al refuted the major conclusions of Schreiner. Behnke et al concluded that the effects reported earlier by Schreiner are irreproducible and result solely from hydrostatic pressure, i.e., that no effects of noble gases upon enzymes are demonstrable. "Enzyme-Catalyzed Reactions as Influenced by Inert Gases at High Pressures." J. Food Sci. 34:370-375.
In essence, the studies of Schreiner were based upon the hypothesis that chemically inert gases compete with oxygen molecules for cellular sites and that oxygen displacement depends upon the ratio of oxygen to inert gas concentrations. This hypothesis was never demonstrated as the greatest observed effects (only inhibitory effects were observed) were observed with nitrous oxide and found to be independent of oxygen partial pressure. Moreover, the inhibition observed was only 1.9% inhibition per atmosphere of added nitrous oxide.
In order to refute the earlier work of Schreiner, Behnke et al independently tested the effect of high hydrostatic pressures upon enzymes, and attempted to reproduce the results obtained by Schreiner. Behnke et al found that increasing gas pressure of nitrogen or argon beyond that necessary to observe a slight inhibition of chymotrypsin, invertase and tyrosinase caused no further increase in inhibition, in direct contrast to the finding of Schreiner.
The findings of Behnke et al can be explained by simple initial hydrostatic inhibition, which is released upon stabilization of pressure. Clearly, the findings cannot be explained by the chemical-O.sub.2 /inert gas interdependence as proposed by Schreiner. Behnke et al concluded that high pressure inert gases inhibit tyrosinase in non-fluid (i.e., gelatin) systems by decreasing oxygen availability, rather than by physically altering the enzyme. This conclusion is in direct contrast to the findings of Schreiner.
In addition to the refutation by Behnke et al, the results reported by Schreiner are difficult, if not impossible, to interpret for other reasons as well.
First, all analyses were performed at very high pressure, and were not controlled for hydrostatic pressure effects.
Second, in many instances, no significant differences were observed between the various noble gases, nor between the noble gases and nitrogen.
Third, knowledge of enzyme mode of action and inhibition was very poor at the time of these studies, as were the purities of enzymes used. It is impossible to be certain that confounding enzyme activities were not present or that measurements were made with a degree of resolution sufficient to rank different gases as to effectiveness. Further, any specific mode of action could only be set forth as an untestable hypothesis.
Fourth, solubility differences between the various gases were not controlled, nor considered in the result.
Fifth, all tests were conducted using high pressures of inert gases superimposed upon 1 atmosphere of air, thus providing inadequate control of oxygen tension.
Sixth, all gas effects reported are only inhibitions.
Seventh, not all of the procedures in the work have been fully described, and may not have been experimentally controlled. Further, long delays after initiation of the enzyme reaction precluded following the entire course of reaction, with resultant loss of the highest readable rates of change.
Eighth, the reported data ranges have high variability based upon a small number of observations, thus precluding significance.
Ninth, the levels of inhibition observed are very small even at high pressures.
Tenth, studies reporting a dependence upon enzyme concentration do not report significant usable figures.
Eleventh, all reports of inhibitory potential of inert gases at low pressures, i.e., &lt;2 atm., are postulated based upon extrapolated lines from high pressure measurements, not actual data.
Finally, it is worthy of reiterating that the results of Behnke et al clearly contradict those reported by Schreiner in several crucial respects, mainly that high pressure effects are small and that hydrostatic effects, which were not controlled by Schreiner, are the primary cause of the incorrect conclusions made in those studies.
Additionally, although it was reported by Sandhoff et al, FEBS Letters, vol. 62, no. 3 (March, 1976) that xenon, nitrous oxide and halothane enhance the activity of particulate sialidase, these results are questionable due to the highly impure enzymes used in this study and are probably due to inhibitory oxidases in the particles.
To summarize the above patents and publications and to mention others related thereto, the following is noted.
Behnke et al (1969), disclose that enzyme-catalyzed reactions are influenced by inert gases at high pressures. J. Food Sci. 34: 370-375.
Schreiner et al (1967), describe inert gas interactions and effects on enzymatically, active proteins. Abstract No. 2209. Fed. Proc. 26:650.
Schreiner, H. R. 1964, Technical Report, describes the physiological effects of argon, helium and the rare gases. Contract Nonr 4115 (00), NR: 102-597. Office of Naval Research, Washington, D.C.
Schreiner, H. R. 1965, Technical Report, describes the physiological effects of argon, helium and the rare gases. Contract Nonr 4115 (00), NR: 102-597. Office of Naval Research, Washington, D.C.
Schreiner, H. R. 1966, Technical Report, describes the physiological effects of argon, helium and the rare gases. Contract Nonr 4115 (00), NR: 102-597. Office of Naval Research, Washington, D.C.
Doebbler, G. F. et al, Fed. Proc. Vol.26, p. 650 (1967) describes the effect of pressure or of reduced oxygen tension upon several different enzymes using the gases Kr, Xe, SF.sub.6, N.sub.2 0, He, Ne, Ar and N.sub.2. All gases were considered equal in their effect.
Colten et al, Undersea Biomed Res. 17(4), 297-304 (1990) describes the combined effect of helium and oxygen with high pressure upon the enzyme glutamate decarboxylase. Notably, only the hyperbaric inhibitory effect of both helium and oxygen and the chemical inhibitory effect of oxygen was noted.
Nevertheless, at present, it is known that enzyme activities can be inhibited in several ways. For example, many enzymes can be inhibited by specific poisons that may be structurally related to their normal substrates. Alternatively, many different reagents are known to be specific inactivators of target enzymes. These reagents generally cause chemical modification at the active site of the enzyme to induce loss of catalytic activity, active-site-directed irreversible inactivation or affinity labeling. See Enzymatic Reaction Mechanisms by C. Walsh (W. H. Freeman & Co., 1979). Alternatively, certain multi-enzyme sequences are known to be regulated by particular enzymes known as regulatory or allosteric enzymes. See Bioenergetics, by A. L. Leninger (Benjamin/Cummings Publishing Co., 1973).
Pectinesterase (EC 3.1.1.11), endo-polygalacturonase (EC 3.2.1.15) and endo-pectin lyase (ED 4.2.2.10) are important pectic enzymes in fruit processing. Pectinesterase de-esterifies pectins producing methanol and pectic acid. PGA and pectin lyase are depolymerases which split glycosidic linkages in their preferred substrates. PGA hydrolyzes low esterified pectins. The combined action of PGA and pectinesterase can also depolymerize high methoxyl pectins.
These enzymes are used extensively in fruit processing, and are the critical determinants of ripening onset and rate in fruit storage and transport. Industrial applications may thus depend upon either externally applied enzymes or upon the enzymes naturally occurring in the product. Further, these enzymes have extremely widespread utility and the function and uses thereof described below are only for purposes of illustration and are not intended to be limitative.
For example, in various processing methods, such as the production of apple juice, pectinases are added to extracted juice to facilitate filtration and prevent gelling in concentrated juice, they are added to the pulp to improve press yield and they are added to liquify pulp.
The processed fruits may be further processed into jams, jellies, dried and rolled food products, pastes and many other products.
Fruit nectars have a high content of fruit ingredients, sugar and sometimes acid. An important factor of cloudy nectars (apricot, mango) is their cloudy stability. Pectic enzymes are used in stabilizing cloudy nectars. Polygalacturonase with added fungal Pectinesterase and polygalacturonase with added exo-arabanase or pure pectin lyase stabilize the cloud, prevent gelling and break cell walls.
Generally, in the ripening of fruit, several enzymes are involved in the degradation of cell walls. For example, PE (pectinesterase), PGA (polygalacturonase), PL (pectin lyase), PAL (pectic acid lyase) and changes in cell walls. These changes may be summarized below in Table 1.
Tech. effects Changes in cell walls Active Enz. Firming: Saponification of cell wall PE (+Ca.sup.+ +) pectin Softening: Limited degradation cell PGA, PL, PAL wall pectin Maceration: Limited degradation middle lamella pectin organized tissue.fwdarw.cell suspension Disintegration: Solubilization cell wall PGA + PE, and/or juice release pectins and associated PL + cloud stabiliz. arabinans/galactans, cell hemicellulases wall fragmentation (arabanases, Liquefaction: Solubilization of all cell galactanases) wall polysaccharides C + PE + PGA and/or PL Saccharification: Degradation of solubilized Hemicellulases PS fragments to mono- Oligomerases E .times. saccharides O - carbohydrases Glycosidases Cloudy juices: inhibit. native cloud destabiliz. Saponification PE clarification soluble/insoluble pectin PE + PGA, PL Depolymerization soluble +insoluble pectin n reduction PE: Pectinesterase PGA: Polygalacturonase PL: Pectin lyase PAL: Pectic acid lyase C: Cellulase
The above cell wall changes are fundamentally important in any fruit processing and ripening processes. Polygalacturonase:
Ripening of tomatoes: found in very low levels in green tomatoes, higher (600 x's) concentrations in ripe tomatoes. PA1 Ripening of peaches: PGA activity found at the onset of ripening and increases sharply during ripening. PA1 Ripening in other fruits: PGA activity is present in pears during the cell division stage, decreases during enlargement stage and markedly increases during ripening. PGA found in Papaya, ripe avocados, dates, ripe apples and mangoes. Also found in cucumbers. PA1 In other plant tissue: carrot roots, citrus leaf explants, sealings of: corn, beans, oats and peas (also found in their stem and leaf tissue). (these are just a few examples).
At present, control of pectinesterases, pectinases, and polygalacturonases is not possible, except by manipulation of physical conditions and enzyme level, applied externally. However, an additional and more reliable means of control would be extremely useful to fruit processors, growers, and shippers.